We propose to study the mechanisms of oxygenase catalyzed O2 activation and insertion reactions. We have purified, characterized, and, in many cases, crystallized a group of Fe-containing intra- and extra- diol catecholic dioxygenases and gentisate 1,2 dioxygenase (1,2 GTD) which appear to be good systems with which to investigate all recognized types of oxygen activation chemistry. These enzymes catalyze cleavage of O2 and insertion of both oxygen atoms into the aromatic ring of their organic substrates, resulting in ring opening. The substrates of these enzymes serve as focal points in pathways of bacterial degradation of aromatic compounds, thus they are of substantial environmental significance. Similar enzymes catalyze essential steps in mammalian biosynthetic pathways. Past studies have lead to the development of models for the mechanisms of both classes of catecholic dioxygenases. It is proposed that intradiol dioxygenases activate substrate for attack by 02, while extradiol dioxygenases activate 02 for attack on the substrate. We now plan to test and refine these models, and to develop a similar mechanistic model for 1,2 GTD. The work will involve studies in 4 areas: structure, kinetics, spectroscopy and synthetic analogs. We have recently obtained the first X-ray crystal structure of a dioxygenase (Protocatechuate 3,4 dioxygenase) which will serve as the basis for the structural studies. Similar crystallographic studies have been initiated for the other dioxygenases. Complementary molecular genetic studies are planned for all of the enzymes. Stopped flow and stopped freeze techniques will be used to investigate the binding of slow substrates and the formation of oxy- intermediates. The structure of the Fe environment of these intermediates will be investigated using optical, EPR, and Mossbauer spectroscopies. EXAFS, NMR, Resonance Raman, and MCD spectroscopies will also be used to study the native enzymes, stable complexes, and intermediates in the reaction cycles. Analogs for the substrates and O2 will be labeled with 13C, and 15N to study their interaction with the Fe through EPR-detected hyperfine interaction. Transition state analogs based on the proposed mechanisms will also be synthesized. These studies will be complemented by coordinated and/or collaborative studies of two novel monooxygenases which catalyze oxidation of methane and ammonia. This work should yield fundamental information about the chemistry of oxygenases, O2 and iron.